Women with breast carcinoma tend to have elevated serum levels of a molecular antigenic determinant referred to as the DF3 antigen (DF3). This provides the basis for a currently used diagnostic assay in which samples of a woman's serum are reacted with antibodies that bind specifically to DF3 (anti-DF3 antibodies; D. F. Hayes et al, J. Clin. Oncol. 4, 1542-1550; D. F. Hayes et al, J. Clin. Invest. 75, 1671-1678, 1985). The current invention relates to genetically engineered molecules that carry an immunologically active portion of the DF3 antigen; i.e., one that reacts with anti-DF3 antibodies. The molecules can be used to improve the reproducibility of the diagnostic assay. They can also be used as the basis for an alternative, more sensitive assay. In a related invention, individual women can be categorized as to their genetic material that determines the structure of DF3.
It has been demonstrated that naturally occurring DF3, that occurring in human breast carcinoma cells or the plasma of patients with breast cancer, is a member of a family of related but not identical high-molecular weight tumor-associated antigens (M. Abe et al, J. Immunol. 139, 257-261, 1987). Naturally occurring DF3 has been partially characterized as a high molecular weight mucin-like glycoprotein (H. Sekine et al., J. Immunol 135, 3610-3615, 1985; M. Abe et al., J. Cell Physiol. 126, 126-132, 1986), a molecule with both a polypeptide component and a carbohydrate component. The polypeptide component is comprised of one or more chains, each consisting of amino acids linked end-to-end in a specific sequence. On the average, it accounts for about 15 percent of the DF3 molecule, there being batch-to-batch variability and, within each batch, molecule-to-molecule variability, in the ratio of polypeptide to carbohydrate. As a result DF3 antigen is a collection of closely related but not necessarily identical molecules having the common property that they react with anti-DF3 antibodies. In human MCF-7 breast carcinoma cells, the antigen consists of two distinct glycoproteins with molecular weights in the range of 330 and 450 kilodaltons (kd), respectively (H. Sekine et al., J. Immunol 135, 3610-3615, 1985; M. Abe et al., J. Cell Physiol. 126, 126-132, 1986). DF3 antigen that circulates in the plasma of patients with breast cancer also has molecular weights ranging from approximately 300 to 450 kd (D. Hayes et al J. Clin. Invest. 75, 1671-1678, 1985).
In the currently used diagnostic assay for DF3, interpretation of the results requires that controls involving known amounts of DF3 be run. DF3 isolated from extracts of carcinoma cells is used to calibrate the assay. However, because of the above noted variability in the structure of DF3 antigen from molecule to molecule and from batch to batch, it would be advantageous to have a method of preparing a more reproducible version of the antigen. Improved reproducibility would be achieved if a carbohydrate-free polypeptide, capable of reacting with anti-DF3 antibody could be prepared. Previous work with the naturally occurring version had suggested that the carbohydrate portion of DF3 was essential for reaction with the anti-DF3 antibody. Nevertheless, in the current invention, synthesis of carbohydrate-free polypeptides with an antigenic determinant capable of reacting with anti-DF3 antibody (DF3 polypeptides), was achieved. Furthermore, these DF3 polypeptides can be synthesized in bacteria, which are expected to provide a less costly means of producing it.
The ability to synthesize an antigenically active polypeptide in bacteria also provides the basis for an alternative, potentially superior, means of detecting DF3 in human sera. In bacteria, the polypeptide can be synthesized with a higher specific radioactivity than is possible in human cells. It can then be used in a competition assay, one where anti-DF3 antibodies are allowed to react with a mixture of radioactive DF3 polypeptide that was synthesized in bacteria and nonradioactive antigen from the person's serum. This type of assay has been used with the carcinoembryonic antigen (CEA; See, for example, Feb. 1983 package insert for Carcinoembryonic Antigen Radioimmunoassay, Roche Diagnostics, Nutley, N.J. 07110) The nonradioactive antigen will compete with the radioactive DF3 for antibody binding sites, the diminished binding of radioactivity being an index of the amount of DF3 antigen in the person's serum. This type of assay is expected to be able to detect smaller amounts of antigen in a person's serum than the currently used assay can.
In an example of the invention, a DF3 polypeptide was synthesized in the prokaryotic organism, Escherichia coli (E. coli), a bacterium. Although there is still uncertainty as to both the number of polypeptide chains in a naturally occurring DF3 antigen molecule and the size of each chain, it is likely that the synthetic DF3 polypeptide represented less than a complete naturally occurring chain. Incomplete synthesis of an antigen polypeptide chain is a possible result of the procedure used to initially isolate DNA coding for an antigenically active site. First, messenger RNA (mRNA) was isolated from human breast carcinoma cells, which are known to synthesize the antigen. DNA copies of the mRNA were then made. (Failure to isolate intact mRNA molecules or synthesize complete DNA copies are two possible reasons why incomplete synthesis of an antigen chain is ultimately achieved.) Each DNA fragment was then attached to the DNA of a bacterial virus such that, if the fragment contained the ability to direct the synthesis of human DF3 polypeptide, that peptide would be expressed as part of a fused polypeptide also containing the bacterial polypeptide, beta galactosidase. The resulting population of DNA molecules was distributed among a very large number of bacterial cells by a transfection process. At a subsequent step, each bacterial cell was tested for the ability to direct the synthesis of a polypeptide that would react with anti-DF3 antibody. Prior to completing the test, there was uncertainty as to whether the procedure employed would be successful in generating bacteria capable of making an antigenically active polypeptide: Not only would the polypeptide lack the carbohydrate portion it has in humans, there was an excellent chance that it would be smaller than its intact human form. As it turned out, several bacteria that produced antigenically active polypeptide were found, the one presented in detail here being typical of the group.
Regardless of the precise relationship of the E. coli produces DF3 polypeptide to the naturally occurring one, the results presented above provide the following picture:
(1) the polypeptide component of DF3 has antigenic activity in the absence of any carbohydrate component;
(2) probably only a portion of the polypeptide (not more than 103 amino acids) is required for antigenic activity; and
(3) the antigenically active portion of the DF3 polypeptide retains its antigenic activity even when part of a polypeptide that is partly comprised of polypeptide sequences naturally foreign to it. As to this latter point, consider the fact that, in E. coli, the DF3 polypeptide was joined to the 116,000 dalton bacterial polypeptide, beta galactosidase. An advantage of this latter property is that the antigenically active site can be made part of a tyrosine-rich polypeptide, and radioactive iodine can be attached to the tyrosine residues, thereby increasing the specific radioactivity of the antigenic probe for use in the competition diagnostic assay.
Electrophoretic mobility patterns of an antigen are a reflection of its structure. The electrophoretic mobility patterns for circulating DF3 antigen are heterogeneous and differ among individuals (D. Hayes et al J. Clin. Invest. 75, 1671-1678, 1985). Subsequent studies in family members have demonstrated that the electrophoretic mobility pattern of plasma DF3 antigen is genetically determined by codominant expression of multiple alleles at a single locus (D. Hayes et al, Blood, 1988, in press).
The aforementioned electrophoretic and genetic studies would not, however, make it obvious how one could detect the changes in DNA structure responsible for the person-to-person variations in DF3 structure. Person-to-person variation in DNA structure of specific genes had been successfully demonstrated for some other genes using the technique of restriction fragment length polymorphism (RFLP). In RFLP analysis, one analyzes the size of DNA fragments that carry a particular gene after controlled digestion (by a restriction endonuclease) of that person's DNA. The technique can be used to categorize individuals genetically and also assist in identifying the individual who is the source of a particular tissue or group of body cells. Applicant used RFLP analysis to investigate the size of DNA fragments that carry the gene for the 103-amino acid antigenically active DF3 polypeptide. He discovered that indeed such RFLP analysis reveals variations in DNA structure that correlated with the variations in size of the circulating antigens. Whether there is a correlation between a particular DNA structure and a predisposition to breast cancer is unknown.
Of interest is a report which describes the isolation of a partial cDNA clone (pMUC10) which codes in part for urinary mucins (PUMs) (D. Swallow et al, Nature 328, 82-84, 1987). The report is of interest because the clone codes for CA1, an antigenic determinant also found on naturally occurring DF3 antigen. Furthermore, an RFLP analysis of human DNA using the pMUC10 probe disclosed EcoRI restriction fragments similar in size to those found in the present study (S. Gendler et al, Proc. Natl. Acad. Sci. U.S. 84: 6060-6064, 1987). Nevertheless it is uncertain to what extent, if any, pMUC10 codes for sequences within the 103-amino acid and 600-amino acid DF3 polypeptides of the present invention. In the first place, Ca1 reacts with a wide range of human tumors (F. Ashall et al, Lancet 2, 1-6, 1982; J. McGee et al, Lancet 2, 7-10, 1982). Additionally, the binding site on the DF3 glycoprotein for anti-Ca1 antibodies is distinct from that for anti-DF3 antibodies (M. Abe et al, J. Immunol. 139: 257-261, 1987). Indeed, none of the aforementioned reports on pMUC10 disclosed that it codes for a determinant recognized by anti-DF3 antibody.